Re-Examining The Role Of Cdc14 In Mitotic Exit
Abstract
Inactivation of cyclin-dependent kinase (Cdk) and reversal of Cdk phosphorylation are universally required for mitotic exit. In budding yeast, the protein phosphatase Cdc14 is thought to perform these essential functions to regulate the ordered series of events of mitotic exit - a requirement not shared by other eukaryotes. Previous studies suggest the ratio of Cdk:Cdc14 activity directs ordered dephosphorylation to coordinate late mitotic events. However, the mechanisms that determine the timing of dephosphorylation for individual Cdk substrates remain unclear. Here, we tested if the timing of mitotic exit is dependent on Cdc14 concentration as predicted by current models. Using an inducible degron system, we found that Cdc14 can be significantly depleted and cells still proceeded through mitotic exit - contrary to current models. Yet, Cdc14 depleted cells had a cytokinesis defect and were not viable. We also tested if intrinsic Cdc14 specificity contributes to the order of Cdk substrate Dephosphorylation. Using quantitative data-indendent LC-MS/MS acquisition, we quantitatively monitored the phosphoproteome in response to Cdc14 expression. Our analysis found that a distinctive subset of Cdk sites were dephosphorylated in response to Cdc14 (strikingly consistent with previous in vitro characterization). Our results suggest that intrinsic specificity of Cdc14 directs ordered dephosphorylation and that Cdc14 is not responsible for bulk Cdk substrate dephosphorylation. Together, these results suggest that protein phosphatase regulation of mitotic exit in S. cerevisiae may be more conserved with other eukaryotes, where Cdc14 is not critical for mitotic exit or bulk Cdk dephosphorylation. Additionally, the novel data-independent LC-MS/MS approach for quantifying phosphoproteome dynamics may be a generally useful tool to globally profile phosphatase or kinase specificities in vivo.
Degree
Ph.D.
Advisors
Hall, Purdue University.
Subject Area
Biochemistry
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